Methods and Results: We established hAMCs. After cardiomyogenic induction in vitro, hAMCs beat spontaneously, and the calculated cardiomyogenic transdifferentiation efficiency was 33%. Transplantation of hAMCs 2 weeks after myocardial infarction improved impaired left ventricular fractional shortening measured by echocardiogram (34±2% [n=8] to 39±2% [n=11]; P<0.05) and decreased myocardial fibrosis area (18±1% [n=9] to 13±1% [n=10]; P<0.05), significantly. Furthermore hAMCs transplanted into the infarcted myocardium of Wistar rats were transdifferentiated into cardiomyocytes in situ and survived for more than 4 weeks after the transplantation without using any immunosuppressant. Immunologic tolerance was caused by the hAMC-derived HLA-G expression, lack of MHC expression of hAMCs, and activation of FOXP3-positive regulatory T cells. Administration of IL-10 or progesterone, which is known to play an important role in feto-maternal tolerance during pregnancy, markedly increased HLA-G expression in hAMCs in vitro and, surprisingly, also increased cardiomyogenic transdifferentiation efficiency in vitro and in vivo.

Conclusions: Because hAMCs have a high ability to transdifferentiate into cardiomyocytes and to acquire immunologic tolerance in vivo, they can be a promising cellular source for allograftable stem cells for cardiac regenerative medicine.

Although embryonic stem cells1 and induced pluripotent stem (iPS) cells2 can be differentiated into cells of various organs, including cardiomyocytes, there are many underlining problems to overcome before clinical applications can be used, eg, tumorigenicity.3 Autografts of iPS cells may not cause immunologic rejection; ironically, however, possible neoplasm formation would cause a serious problem because the neoplasm would not be rejected by the withdrawal of immunosuppressive agents. On the other hand, mesenchymal stem cells (MSCs) have recently been used for clinical application, and their safety and feasibility in cardiac stem cell-based therapy have been demonstrated.4 Thus, MSCs are a more important cellular source for stem cell-based therapy from a practical point of view.

The efficacy of human bone marrow–derived MSCs (BMMSCs) was still limited,5 however, because of low efficiency for cardiomyogenic transdifferentiation.6 We previously reported that non–marrow-derived mesenchymal cells had higher cardiomyogenic transdifferentiation efficiency, eg, menstrual blood–derived mesenchymal cells (MMCs),7 umbilical cord blood–derived mesenchymal stem cells (UCB-MSCs),8 and placental chorionic plate–derived mesenchymal cells (PCPCs).9 These cells are thought to be used by an allograft; therefore, problems of immunologic rejection arise. However, an allograft may be superior to an autograft in several ways. Taking into account the background condition of the patient (eg, metabolic disease or age), stem cells obtained from young and healthy volunteers are expected to have a better efficacy in stem cell therapy.10,11 Previously, mesenchymal cells did not express HLA-DR6–9,12 and are believed to resist immunologic rejection to some extent. Shake et al13 showed that xenografted mesenchymal cells were immunologically tolerated in the host heart. These cells, however, failed to show clear cardiomyogenic differentiation, and the mechanisms of tolerance were not well investigated. Unlike other mesenchymal cells (BMMSC, MMC, UCB-MSC, PCPC), only human amniotic membrane–derived mesenchymal stem cells (hAMCs) do not express the major histocompatibility complex (MHC) class I molecule and may be expected to show immunologic tolerance.

Recently, amniotic membrane–derived cells were reported to have potential for transdifferentiation into cells of various organs. Zhao et al,14 and Miki et al,15 reported evidence of possible cardiomyogenic transdifferentiation ability, but failed to show functioning cardiomyocytes. Fujimoto et al,16 reported significant recovery of cardiac function by the rat amnion-derived cell transplantation in rat myocardial infarction (MI) model, however, they failed to show clear evidence of cardiomyogenic differentiation in vivo. Therefore, in the present study, we attempted to show: (1) the powerful cardiomyogenic transdifferentiation potential of our isolated hAMCs, and the beneficial effect of transplantation of hAMCs on cardiac function in vivo; (2) the induction of immunologic tolerance so that hAMCs can be a powerful allograftable stem cell source without either the administration of immunosuppressive agents or matching of MHC typing; (3) the mechanism of induction of tolerance; and (4) the close relationship between the cardiomyogenic transdifferentiation of mesenchymal cells and the process of immunologic tolerance.

Methods

Human amniotic membrane was collected, with informed consent from individual patients, after delivery of a male neonate. The study was approved by the ethics committee of Keio University School of Medicine. The precise methods for culture have been described previously.9,17 Detail is shown in the Online Data Supplement.

Coculture With Murine Fetal Cardiomyocytes

Cardiomyocytes were obtained from hearts of day-17 mouse fetuses.6–9 The isolated cardiomyocytes were replated at 5×104/cm2 on top of a floating atelocollagen film,7,8 or on the bottom of a culture dish. The human amniotic membrane–derived mesenchymal cell (hAMCs) were infected with enhanced green fluorescent protein (EGFP)-expressing adenovirus.6–9 The hAMCs were harvested and seeded on top of the cardiomyocytes (Figure 1H and 1I; Figure 2A, 2B, and 2H through 2M; Figure 3; Online Figure III) or on the bottom of the atelocollagen film (Figure 2D through 2F; Online Figure III) at 3×103/cm2. For blocking experiments, neutralizing antibodies directed to HLA-G (clone 87G; Exbio) and IL10 (ab22771; Abacam) were added at a concentration of 20 μg/mL every day from 1 day before coculture till day 7. To prove the effect of immunologic reaction to the coculture system, we used the following immunosuppressive agents from one day before the coculture to day 7: FK506 (tacrolimus, 20 ng/mL, F4679, Sigma-Aldrich) and hydrocortisone (1 μg/mL, H0396, Sigma-Aldrich). In some experiment we used EGFP transgenic mouse fetuses (C57BL/618).

Figure 1. Cellular phenotype, surface marker, and expression of cardiomyocyte-specific genes. A, Macroscopic view of amniotic membrane. B, Phase contrast microscopic view of hAMCs. C, The representative growth curves of hAMCs as a function of time after the culture. D, Numbers of collected mesenchymal cells obtained from 1 g of placental tissues at 2 weeks after the start of culture are shown. n indicates number of delivery. E through G, Laser confocal microscopic view of immunocytochemistry of hAMCs in culture with anti-pancytokeratin (green; F) and anti-vimentin (red; G) antibodies. H andI, RT-PCR was performed with PCR primers with specificity for human genes encoding cardiac proteins but not for the corresponding murine genes (Online Table I) before (−, white box) and after (+, black box) the coculture. H, RT-PCR for stem cell markers was performed. hAMCs express Oct-4 and c-kit at the default state. I, Human heart cells and mouse heart cells were used as a positive control and negative control, respectively. Most human cardiac genes were expressed after the coculture. Scale bars: 20 μm (B and G).

Figure 3. Effect of hAMC transplantation on heart function in vivo. A, Representative traces of M-mode echocardiogram from the control MI of nude rats and MI+hAMC transplanted nude rats (MI+hAMC) are shown. The contraction of the left ventricular anterior wall was improved by transplantation of hAMCs (whitearrows). B, The hAMC transplantation significantly increased in measured percentage of left ventricular fractional shortening (%LVEF) at 4 weeks after the first operation. C and D, Representative Masson’s trichrome staining of the heart at the papillary muscle level is shown. The digitized data were measured and calculated in E. The hAMC transplantation significantly decreased in percentage of fibrosis area. Scale bar: 1 mm. F, Merged image of confocal laser microscopic view of immunohistochemistry with anti–cardiac troponin I antibody (Trop-I) (red), DAPI (blue), and EGFP (green). The EGFP-positive hAMC-derived cardiomyocytes were observed at the margin of the MI area of the nude rats. Scale bar: 50 μm.

Immunocytochemistry and Immunohistochemistry

A laser confocal microscope (FV1000, Olympus) was used for immunocytochemical analysis. Samples were stained with anti–cardiac troponin-I antibody or anti–sarcomeric α-actinin antibody and anti–connexin 43 antibody, as described previously.7–9 In the present study, we used anti-FOXP3 antibody (IMGENEX, IMG-5276A), anti–HLA-G antibody (Abcam, ab7758), and anti-hANP antibody (YLEM, MCV928) according to the manufacturer’s recommendation. The cells were isolated and stained immunocytochemically, and then observed by confocal laser microscope. The cardiomyogenic transdifferentiation efficiency was calculated as the fraction of cardiac troponin-I positive cells in the EGFP-positive cells.7–9 Detail is shown in the Online Data Supplement. The methods to evaluate in vitro transdifferentiation potential to the noncardiac organs are provided in the Online Data Supplement.

Reverse Transcriptase–Polymerase Chain Reaction

RT-PCR was done as described previously.6–9 PCR primers were prepared such that they would amplify the human but not the mouse genes. Primers for cardiomyocyte-related genes were used (see also Online Table I).

Western Blot and ELISA

Western blot analysis for hAMCs was done by iBlot dry blotting system (Invitrogen) according to the manufacturer’s recommendation. Proteins were extracted from 1×106 hAMCs, placenta-derived mesenchymal cells, and menstrual blood–derived mesenchymal cells. Cellular lysates were electroblotted and probed using the anti–HLA-G IgG monoclonal antibody (1 μg/mL; EXBIO). Collected images were analyzed by the Image J software (http://rsbweb.nih.gov/ij/). The calculated data were normalized by the data of b-actin. Soluble HLA-G was measured by enzyme-linked immunosorbent assay (ELISA) in plates coated with the captured antibody MEM-G/09 (sHLA-G Kit; Exbio, Czech Republic), according to the manufacturer’s recommendation. JEG3 (GeneTex, GTX14841) was used for the positive control of HLA-G.

Fluorescent In Situ Hybridization

The CEP X/Y DNA Probe Kit (Vysis) was used to determine the proportion of XX and XY cells according to the manufacturer’s recommendation.9 The Alu probe (BIOGENEX, PR-100101) was used according to the manufacturer’s recommendation.

Physiological Analysis

Functional analysis was performed at 1 week after the cardiomyogenic induction. The method of action potential (AP) recording was as previously described.6–9 Alexa 568 was injected into cells via recording microelectrodes to stain the cells and confirm that the AP was generated by EGFP-positive cells (Figure 1B). Contraction of the cells was measured by the video image of EGFP-positive cells as described previously.7–9

hAMC Transplantation in Myocardial Infarction Model In Vivo

Myocardial infarction (MI) was induced in the open chests of anesthetized Wistar rats (8 weeks of age) or of F344 nude rats (Clea Japan Inc) (6 weeks of age), as described previously.7,19 Two weeks after the MI, 1 to 2×106 of EGFP-labeled hAMCs were injected into the myocardium at the border zone of the MI7; they survived without using an immunosuppressive agent. In some experiment EGFP-transgenic mouse18 was uses as recipients. To examine the effect of hAMC transplantation in vivo, we selected nude rats as recipients.7 Two weeks after the first operation, nude rats with MI were randomized in a blind study of the following groups: the control MI group (MI), the MI+hAMC transplanted group (MI+hAMC), and the sham operated group (sham). Randomization occurred immediately before echocardiogram. Immediately before cell transplantation, 2D and M-mode echocardiographic (8.5-MHz linear transducer; EnVisor C, Phillips Medical System, Andover, Mass) images were obtained to assess left ventricular end-diastolic dimension and left ventricular end-systolic dimension at the midpapillary muscle level. Two weeks after the transplantation, a similar echocardiogram was performed again. ECG and left ventricular pressure were measured as previously described.7 Tissue samples were obtained by slicing along the short axis of the left ventricle, for every 1 mm of depth. After Masson’s trichrome staining, the area of fibrosis was digitized from each slice, and then the percentage of fibrosis area in the left ventricular myocardium was calculated, as previously described.7 To test the potential induction of immunologic tolerance, hAMCs were transplanted into the Wistar rats. Hearts and sera were obtained at between 2 days to 56 days after hAMC transplantation, and then analyzed by immunohistochemistry, fluorescent in situ hybridization (FISH), and ELISA experimental methodologies. The survival rate of EGFP-positive cardiomyocytes was calculated by fluorescent microscope (details are shown in the Online Data Supplement). In some experiments, hAMCs were pretreated with 10 ng/mL of IL10 (Sigma I9276) or 10 ng/mL of progesterone (p7556; Sigma-Aldrich) for 2-days before the transplantation to observe the efficacy of survival of hAMCs in vivo. Enzymatically isolated EGFP-positive cardiomyocytes20,21 were selected by glass pipette driven by a manipulator mounted on the inverted fluorescent microscope, then used for the FISH experiment to determine the origin of EGFP-positive transdifferentiated cardiomyocytes. See also the Online Data Supplement.

Statistical Analysis

All data are shown as the mean value ± SE. The difference between two mean values was determined with a Student t test. The difference among more than 3 mean values was determined with one-way ANOVA test or one-way repeated measures ANOVA test and Bonferroni post hoc test. Statistical significance was set at P<0.05.

Results

This study was approved by the institutional ethical committee. With informed consent, human placenta and amniotic membrane were collected after delivery of a male neonate. The amniotic membrane was pealed off from the maternal placenta (Figure 1A) and hAMCs (Figure 1B) were collected by the culture method, as described previously.9,17 The hAMCs proliferated at 18 to 22 population doublings (PDs) (Figure 1C), and experiments were performed on hAMCs at 2 to 9 PDs unless otherwise mentioned. The number of mesenchymal cells at 3 days after the primary culture per 1g tissue samples from the amniotic membrane, umbilical cord, and placenta9 are shown in Figure 1D. This suggests that the amniotic membrane is a rich cellular source of mesenchymal cells. FISH analysis for human chromosome X and Y revealed that 100% of the obtained cells were of male infant origin. Immunocytochemical analysis revealed that hAMCs expressed both pancytokeratin and vimentin, suggesting hAMCs have both epithelial and mesenchymal phenotypes (Figure 1E through EG). Surface marker analysis (Online Figure I) revealed that hAMCs did not express hematopoietic linage markers, eg, CD14, CD34, CD45, CD117, and CD309, and did express mesenchymal linage markers, eg, CD10, CD29, CD44, and CD105. It is noteworthy that hAMCs were positive for SSEA4,22 an embryonic stem cell marker. hAMCs were negative for HLA-ABC, HLA-D, and HLA-DR. The RT-PCR was performed with primers that hybridized with human-specific genes but not with the murine orthologs.6–8 The hAMCs expressed Oct-4 and c-kit (Figure 1H) and did not express Nkx2.5 before cardiomyogenic induction (Figure 1I). Almost all cardiac-specific genes were expressed after cardiomyogenic induction (Figure 1I).

Cardiomyogenic induction of hAMCs was performed by a coculture system with fetal murine cardiomyocytes, as described previously.6–9 EGFP-labeled hAMCs started beating within a few days after the induction and about half of the hAMCs spontaneously contracted in a synchronized manner (Figure 2A); the averaged percentage of fractional shortening (%FS) was 6.2±0.6% (n=8). The recorded AP from EGFP-positive hAMCs (Figure 2B) showed pacemaker-like potential (n=7) and cardiomyocyte-specific long AP duration (Figure 2C). Averaged amplitude was 71.5±2.2 mV, maximal diastolic potential was -52.7±1.9 mV, AP duration at 90% repolarization was 161.6±9.3 ms, and beating cycle length was 1.06±0.1 s (n=7). Cardiomyogenic transdifferentiation could be observed when the murine cardiomyocytes and hAMCs were separately cocultured by the atelocollagen membrane (Figure 2D through 2F; Online Figure III) that is permeable for only small molecules (less than 5,000MW).7,8 In another experiment murine cardiomyocytes were stained with MitoTracker Red (Invitrogen, M7512) and cocultured with hAMCs, then we confirmed that almost all EGFP positive cardiomyocytes were MitoTracker negative (Online Figure II, A through E). On the other hand hAMCs were stained with the MitoTracker Red and cocultured with EGFP-transgenic murine cardiomyocytes, then we confirmed that almost all MitoTracker positive cardiomyocytes were EGFP negative (Online Figure II, F through J). Thus, we concluded that the observed EGFP-positive cardiomyocytes were caused not by cell fusion between murine cardiomyocytes and hAMCs, but by transdifferentiation of hAMCs. Immunocytochemical analysis revealed a clear striation pattern of cardiac troponin-I (Figure 2D), dot-like pattern of human atrial natriuretic peptide (hANP) (Figure 2E), clear striation pattern of α-actinin, and dotted staining of connexin 43 (Figure 2F and Online Figure III). The percentage of cardiac troponin-I positive cells in the EGFP-positive cells was defined by immunocytochemical analysis (Figure 2G through 2M) and calculated to determine the cardiomyogenic transdifferentiation efficiency.7–9 The efficiency was significantly increased up to 33±3% (n=8) by the cocultivation.

The hAMCs were transplanted into the hearts of nude rats with chronic MI, in vivo, and the effect on cardiac function was examined. Echocardiography showed a significant increase in the left ventricular fractional shortening (%LVFS) at 2 weeks after transplantation (Figure 3A and 3B; see also Online Figure IV). The heart section was stained with Masson’s trichrome (Figure 3C and 3D) and the MI area was digitized and measured (Figure 3E). The percentage of fibrosis area was significantly decreased by hAMC transplantation (MI n=8, MI+hAMC n=11, P<0.05). The EGFP-positive cells of hAMCs (Figure 3F) observed at the MI area expressed a clear striation staining pattern of cardiac troponin-I, suggesting in situ cardiomyogenic transdifferentiation ability for hAMCs. The rate of survived EGFP-positive cardiomyocytes was 1.125±0.470% (n=6).

We tested whether xenografted hAMCs may be immunologically tolerated to survive for more than 2 weeks and differentiate into cardiomyocytes in situ. Isolated hAMCs were injected into the MI zone of female Wistar rats (Figure 4A, inset, white arrow). A significant number of EGFP-labeled rod-shaped cardiomyocytes were observed (Figure 4A and 4B), even 2 weeks after the transplantation (at least 80 days; Online Figure V). Immunohistochemistry revealed that they were positive for sarcomeric α-actinin, connexin 43, and cardiac troponin-I (Figure 4C and 4D; Online Figures VI and VII). Host hearts were enzymatically isolated (Figure 4E and 4F), then EGFP-positive cardiomyocytes were selected (Figure 4G and 4H). FISH analysis to detect the human-Y and the rat-X chromosome revealed that the EGFP-negative cardiomyocytes express the rat-X chromosome and no human-Y chromosome (Figure 4I and 4K), whereas the EGFP-positive cardiomyocytes express the human-Y chromosome and no rat-X chromosome (Figure 4J and 4L). FISH analysis to detect Alu,23 which is a human-specific short interspersed repetitive element, revealed that the EGFP-negative cardiomyocytes were negative for Alu (Figure 4M and 4P), whereas the EGFP-positive cardiomyocytes were positive for Alu (Figure 4O and 4Q). From these findings, we concluded that neither cell fusion nor nuclear fusion was the major cause of generation of EGFP-positive cardiomyocytes, but that the hAMCs transdifferentiated into cardiomyocytes and were immunologically tolerated, surviving more than 80 days in situ. In some experiment non–EGFP-labeled hAMCs was transplanted into the MI zone of EGFP-transgenic mouse, and observed EGFP-negative sarcomeric α-actinin positive hAMCs derived cardiomyocytes was observed (Online Figure VIII).

Before the transplantation, HLA-G was consistently detected in hAMCs by western blot analysis, and was also detected in mesenchymal cells obtained from other placenta-related organs (Figure 5A). After transplantation, however, HLA-G was detected only inconsistently in situ. No membrane-binding isoform of HLA-G was detected in the surviving hAMC-derived cardiomyocytes in Wistar rat hearts (Figure 5B) by immunohistochemical analysis. There was no correlation between continuous secretion of the soluble HLA-G in the sera (ELISA) and survival of hAMC-derived cardiomyocytes (Figure 5C). On the other hand, adjacent to the surviving hAMC-derived cardiomyocytes, FOXP3-positive regulatory T cells were constantly detected by immunohistochemistry (Figure 5D and 5E; Online Figure IX), whereas they were not detected in control myocardium. The effects of IL10 or progesterone on the HLA-G expression in hAMCs were examined. Western blot analysis showed that IL10 and progesterone increased the HLA-G expression (Figure 5F). Concordantly, pretreatment with IL10 or progesterone before the hAMC transplantation significantly increased the incidence of survival of EGFP-positive hAMC-derived cardiomyocytes in vivo (Figure 5G).

Figure 5. Role of HLA-G and regulatory T cells in immunologic tolerance of xenografted hAMCs. A, Western blot analysis of HLA-G in the mesenchymal cells obtained from amniotic membrane (hAMCs), umbilical cord, and placenta. Densitometry analysis of HLA-G, normalized by β-actin. B, Laser confocal microscopic view of immunohistochemistry of host myocardium (2 weeks after transplantation) with anti–HLA-G antibody (red). The transdifferentiated EGFP-positive cardiomyocytes did not show any membrane biding isoform of HLA-G in situ. C, Concentration of the soluble form of HLA-G (sHLA-G) in the sera of hAMC transplanted Wistar rats detected by ELISA as a function of time in weeks after the transplantation is shown. There is no correlation between the survival of EGFP-positive cardiomyocytes and sHLA-G concentration. D, Immunohistochemistry with anti-FOXP3 (red) antibody. E, Expansion of area within the white box in D (see also Online Figure IX). A significant number of FOXP3-positive regulatory T cells have migrated beside the hAMC-derived cardiomyocytes. F, The effect of agents related to fetomaternal immunologic interaction on the HLA-G expression in the hAMCs was tested by Western blot analysis. Densitometric data were normalized by b-actin. Both IL10 and progesterone (prog) markedly increased the HLA-G expression in hAMCs. Concordant with F, pretreatment with progesterone and IL10 significantly increased the survival rate of xenografted hAMC-derived cardiomyocytes in the Wistar rat heart (G). Scale bars: 50 μm (B,D, andE).

Furthermore, the effect of IL10 or progesterone on the cardiomyogenic transdifferentiation efficiency of hAMCs was examined. IL10 or progesterone was administrated to the hAMCs (PDs=13) before and/or after the cardiomyogenic induction, then cardiomyogenic transdifferentiation efficiency was measured (Figure 6A and 6B). Surprisingly, both IL10 and progesterone significantly improved cardiomyogenic transdifferentiation efficiency in vitro. It is notable that pretreatment with IL10 or progesterone before cardiomyogenic induction is essential for this increase in cardiomyogenic transdifferentiation efficiency. Administration of FK506 and hydrocortisone significantly attenuated the IL10-induced increase in cardiomyogenic transdifferentiation efficiency (Figure 6C) in vitro. Moreover, the effect of IL10 was completely blocked by either anti IL10 antibody or anti HLA-G antibody administration (Figure 6D).

Figure 6. The effect of IL10, progesterone, and immunosuppressive agents on cardiomyogenic transdifferentiation efficiency of hAMCs. The effect of IL10 (A) and progesterone (prog) (B) on the cardiomyogenic transdifferentiation efficiency was measured. These agents were administered 2 days before the cocultivation (pretreat) and/or after the cocultivation (posttreat), and the conditions of administration are denoted below each column. Pretreatment with IL10 markedly increased the efficiency of cardiomyogenesis; progesterone, modestly, but significantly, increased the efficiency. C, IL10-induced increase in the cardiomyogenic transdifferentiation efficiency was significantly attenuated by the administration with FK506 or hydrocortisone (COR). D, IL10-induced increase in cardiomyogenic transdifferentiation efficiency was completely blocked by the administration of anti HLA-G antibody and anti-IL10 antibody.

Discussion

We isolated hAMCs in the present study. Our isolated hAMCs can be transdifferentiated into cardiomyocytes in vitro and vivo, without using any epigenetic agent or gene transfer. The cardiomyogenic transdifferentiation efficiency of hAMCs was significantly higher than that of marrow-derived mesenchymal stem cells. Furthermore, xenografted hAMCs transdifferentiated into cardiomyocytes and survived more than 2 weeks (observed up to 80 days; Online Figure V); this suggests hAMCs were tolerated in situ. Immunologic tolerance and cardiomyogenic transdifferentiation of hAMCs were significantly increased by pretreatment with IL10 or progesterone. From these findings, we concluded hAMCs can be a ready-to-use allograftable cellular source for cardiac stem cell therapy.

Highly Cardiomyogenic Transdifferentiation of hAMCs

The cardiomyogenic transdifferentiation efficiency of hAMCs at PD4 was calculated as 33%, which is higher than that of PCPCs.9 Moreover, hAMCs express OCT-4 and SSEA4, a stem cell marker, and have a potential to differentiate into endodermal, mesodermal, and ectodermal lineage (Online Figure X) to some extent. These findings indicate that hAMCs have a ability to transdifferentiate into cells of various organs in comparison to other human somatic stem cells. This potential may contribute to the high cardiomyogenic transdifferentiation ability of hAMCs. The surface marker analysis clearly showed hAMCs have a mesenchymal phenotype. Despite a lack of expression of Nkx 2.5, a cardiac homeobox gene for cardiac differentiation, several cardiomyocyte-specific genes were expressed at the default state of hAMCs. Histological and physiological examinations revealed that hAMCs transdifferentiated into matured and physiologically functioning cardiomyocytes in vitro and in vivo. Moreover, transplantation of hAMCs improved cardiac functions and reduced the area of MI in vivo. The hAMCs-derived cardiomyocytes may play a role in improvement of cardiac function; however, antiapoptotic effect24 of hAMCs may play a significant role in the present study. In the present study, neovascularization25,26 may not play a major role in the improvement because hAMCs did not affect the capillary density (Online Figure XI).

Evidence of Tolerance

Tolerance is extremely important for clinical application of hAMCs, because we can use enormous numbers of hAMCs obtained from every delivery without establishing a stem cell bank system to match host and donor HLA-types. It is also notable that massive numbers of hAMC-derived cardiomyocytes were tolerated and survived in the xenografted host heart without using an immunosuppressant.

It is common that either allografted or xenografted cells could not survive for more than 2 weeks in the immunocompetent hosts because of rejection by the host’s immune system. Therefore, massive survival of transdifferentiated EGFP-positive hAMCs in the Wistar rat heart for more than 2 weeks strongly suggests tolerance. Two independent FISH analysis clearly support the conclusion that the isolated EGFP-positive cardiomyocytes are of hAMC origin (have human nuclei) and are evidence of tolerance in the host heart.

The Mechanism of Tolerance

Because placenta and amniotic membrane are known to play an important role in avoiding maternal immunologic rejection against the fetal tissue bearing paternal alloantigens during normal gestation, it may be possible that the engrafted hAMCs were immunologically tolerated in the host heart. In comparison to other mesenchymal cells, hAMCs express lower MHC antigens; this might have importance for inducing tolerance in the host, because xenografted MMCs,7 which express HLA-G and HLA-ABC, were completely rejected (n=4, data not shown). The expression pattern of HLAs suggests that hAMCs are resistant to MHC-dependent rejection mediated by T cell immune systems, but are known to be rejected by substitutive mechanisms. Cells that do not express the MHC molecule may be recognized as missing self cells, and may be attacked by natural killer cells.27

The nonclassic MHC class I antigen HLA-G expressed on the extravillous cytotrophoblast cells at the fetomaternal interface, is thought to play a major role in protecting the fetus from maternal rejection by natural killer cells.28 Furthermore, HLA-G blocks the immunologic response of natural killer cells29 and induces regulatory T cells,30 which play an important role in immunologic tolerance.31–33 In the present study, despite the fact that hAMCs expressed HLA-G in vitro, there is no correlation between the survival of EGFP-positive cardiomyocytes and continuous secretion of sHLA-G/HLA-G in Wistar rats in vivo. From these findings we speculated that HLA-G might play a role in the initial process of tolerance; however, it may not play a major role in tolerance maintenance.

A previous report showed that HLA-G–induced regulatory T cells, defined as FOXP3 positive lymphocytes,30 also play a significant role in tolerance maintenance. Emergence and mobilization of FOXP3-positive lymphocytes beside the survived EGFP-positive cardiomyocytes in the present study strongly suggest that the regulatory T cell also plays an important role in maintenance of immunologic tolerance. However, it is difficult to verify this hypothesis by observing survival of hAMC-derived cardiomyocytes to evaluate tolerance, because the blockade of HLA-G must inhibit cardiomyogenic transdifferentiation efficiency in vivo. Because both tolerance and transdifferentiation efficiency increase the number of hAMC-derived cardiomyocytes in vivo, it is difficult to demonstrate direct evidence for the role of HLA-G in tolerance by simply determining the number of hAMC-derived cardiomyocytes. Further experimentation should be performed.

IL10, known as an immunosuppressive cytokine, is produced by regulatory T cells and type 2 helper T cells.34,35 Progesterone and IL10, which are known to play an important role in causing fetomaternal immunologic tolerance and maintenance of normal pregnancy,32,36,37 dramatically increase the HLA-G secretion from hAMCs. Concordant with the degree of HLA-G expression, the pretreatment of hAMCs with IL10 significantly increased the survival rate of EGFP-positive cardiomyocytes in Wistar rat hearts. This also suggested the major role of HLA-G in immunologic tolerance in the present study.

Relation Between the Immunologic Reaction and Cardiomyogenic Transdifferentiation of hAMCs

The mechanism of cardiac transdifferentiation of hAMCs is still undetermined; however, it is notable that the immunosuppressive cytokine IL10, or progesterone, dramatically increased the cardiomyogenic transdifferentiation efficiency, whereas FK506 or hydrocortisone attenuated the efficiency. This finding was also associated with the fact that mesenchymal cells, having significant cardiomyogenic transdifferentiation ability, can be obtained from gestation-related organs, ie, umbilical cord blood,8 uterine endometrium,7 menstrual blood,7 placenta,9 and amniotic membrane, and gestation is one of the best circumstances for immunologic tolerance.

Because pretreatment of hAMCs with IL10 before coculture was essential for the increase in cardiomyogenic transdifferentiation efficiency, the main effect of IL10 should be on the hAMCs. IL10 increased the HLA-G expression in hAMCs and the effect on the cardiomyogenic transdifferentiation was blocked by the administration of anti-IL10 antibody or anti–HLA-G antibody, suggesting IL10-induced HLA-G expression plays a pivotal role in increasing the efficiency of cardiomyogenic transdifferentiation. Administration of IL10 without coculture did not cause any cardiomyogenic transdifferentiation in vitro; therefore, the HLA-G–dependent increase in the cardiomyogenesis required the feeder cardiomyocyte culture. The fact that treatment with FK506 or hydrocortisone significantly attenuated the cardiomyogenic transdifferentiation efficiency strongly suggests that inflammation activity in feeder cultures plays a pivotal role in cardiomyogenic transdifferentiation of mesenchymal cells. The different responses in cardiomyogenesis by the same immunologic suppressant may be caused by the different mechanisms of the agents. FK506 and hydrocortisone suppress every immunologic process, whereas HLA-G is known to suppress natural killer cells38 and activate immunosuppressive regulatory T cells.30 The process of HLA-G–dependent induction of immunologic reaction may be a clue to understanding cardiomyogenic transdifferentiation of hAMCs in vitro and vivo. Further experimentation should be performed.

Clinical Contributions

Amniotic membrane can be obtained at every delivery. Because it is usually considered as medical waste, it is an easily accessible cellular source without ethical problems. Our established hAMCs can transdifferentiate into cells of various organs, especially into functioning cardiomyocytes. For safety concerns (ie, neoplasm formation), hAMCs may be superior to the iPS cells, because hAMCs do not require any genetic or epigenetic modifications. We did not observed tumor and/or teratoma formation (also see the Online Data Supplement) in the present study. The noncardiomyogenic transdifferentiation efficiency of hAMCs in vitro was low and hAMCs are subject to contact inhibition and anchorage dependence during multiplication. These cellular characteristics might contribute to the low incidence of neoplasm formation of hAMCs. Regarding immunologic rejection, hAMCs may have an equivalent potential to iPS cells. Because pretreatment with IL10 significantly increased cardiomyogenic transdifferentiation efficiency and immunologic tolerance, we may be able to use hAMCs as an allograftable stem cell source for cardiac stem cell therapy. Compared to the other human mesenchymal cells,6–9 only hAMCs can be immunologically tolerated and transdifferentiate into cardiomyocytes in vivo. Because they can be used as an allograft, hAMCs can be used for clinical patients immediately without establishing a stem cell bank system.

Acknowledgments

We thank S. Hiroi for technical advice in the FISH experiment. EGFP transgenic mice were obtained by courtesy of Dr M. Okabe.

Sources of Funding

The research of H.T. and Y.I. was partially supported by a grant from the Ministry of Education, Science and Culture, Japan. A part of this work was undertaken at the Keio Integrated Medical Research Center.

There is no definitive ready-to-use cellular source for cardiac stem cell therapy. Our present study showed that xenografted human amniotic membrane–derived mesenchymal stem cells (hAMCs) transdifferentiated into cardiomyocytes and were tolerated >80 days in situ. This finding suggested that hAMCs could be an ideal allograftable cardiac stem cell source. Because hAMCs do not cause immunologic rejection in the host, we do not need to adjust donor–host immunologic matching of human leukocyte antigens. Therefore, we can use freshly isolated hAMCs immediately in allogenic combination without establishing stem cell bank systems. Moreover, the precise mechanism of cardiomyogenic transdifferentiation is unclear. We showed close relationships between the mechanisms of cardiomyogenic transdifferentiation and the process of causing immunologic tolerance. This may be a key to understanding of the mechanism of cardiomyogenic transdifferentiation. Consequently, we may be able to manipulate the cardiomyogenic transdifferentiation efficiency. Our findings are important to advance clinical cardiac stem cell therapy.